Understanding nanocarrier pharmacokinetics is crucial for the emerging nanopharmacy, which highly demands noninvasive and real-time visualization of the in vivo dynamics of nanocarriers. To this end, we have developed a 2-photon excitation and time-resolved (TPE-TR) bioimaging apparatus for the analysis of the spatial distribution and temporal evolution of nanocarriers in living model animals. The specific polymeric nanocarrier, Eu@pmma-maa doped with Eu-complexes luminescing in long persistence at ∼615 nm upon near-infrared 2-photon excitation, allows the complete rejection of tissue autofluorescence by selective luminescence detection. This together with a unique beam shaping scheme for homogeneous line excitation, a delicate timing strategy for single-shot line scanning, and an equal optical path design for in-plane scan endows the TPE-TR apparatus with the following prominent features: an imaging depth of ∼10 mm, a field of view (FOV) of 32 × 32 mm2 along with a horizontal resolution of ∼60 μm, a sub-10 s frame time, and negligible laser heating effect. In addition, a combination of the in-plane line scan with the 3D scan of a model animal offers the convenience for examining an interested FOV with a millimeter vertical resolution. Application of TPE-TR bioimaging to a living mouse reveals rich information on the dynamics of nanocarriers including the spatial distribution and temporal evolution and the kinetics of domains of interest. The noninvasive TPE-TR bioimaging instrumentation with a wide FOV and a large imaging depth will find applications in the pharmaceutical development of nanocarriers and relevant research fields.